The gonadal steroids 17 Beta-estradiol (E) and testosterone (T) modulate luteinizing hormone (LH) secretion by affecting the pituitary response to LH releasing hormone (LHRH). Steroids also affect the response to Ca++, which may be a second messenger for LHRH. The long-term objective of this proposal is to elucidate the biochemical mechanisms underlying these steroid actions. This will be done by identifying specific intracellular processes that are affected by steroids and showing that there is a correlation between steroid-induced biochemical changes and steroid effects on LH secretion. The studies will employ enzymatically dispersed anterior pituitary cells derived from 21 day old female rats. In some cases, gonadotrophs will be purified from these heterogeneous cell populations, using velocity sedimentation at unit gravity and cell affinity chromatography. Cells will be pretreated for 48 h with physiological concentrations of T (10 nM) or E (1 nM) and then used to examine steroid effects on the following specific processes: (i) LHRH- and Ca++ stimulated 45Ca++ efflux from perifused cells; (ii) LHRH- and Ca++ induced intracellular calmodulin redistribution, using cell fractionation techniques and a calmodulin radioimmunoassay; (iii) LHRH- and Ca++ stimulated phosphorylation of endogenous proteins, using two-dimensional polyacrylamide gel electrophoresis and autoradiography to quantify 32p incorporation into specific proteins; and (iv) the two phases of the biphasic LH secretory response to LHRH and Ca++, using perifused cells and an LH radioimmunoassay. These studies will determine whether steroids affect intracellular phenomena such as membrane Ca++ permeability, Ca++ mobilization, calmodulin levels or intracellular distribution, Ca++ dependent protein kinases or their substrates, oxocytosis, or secretory granule movement. These effects will be correlated with steroid effects on LH secretion by showing that both can be blocked by steroid antagonists. Such results will help to clarify the mechanisms underlying steroid modulation of LH secretion. This information is of great importance to the understanding of the basic processes involved in both steroid action and LH secretion, and has potential applications to the resolution of problems of both fertility and sterility.